The human CD2 molecule is found on virtually all T cells and thymocytes as well as NK cells and binds to the surface glycoprotein CD58 which is present on many cell types including antigen presenting cells (APCs) (Bierer et al., 1989; Krensky et al., 1983; Moingeon et al., 1989a; Sanchez-Madrid et al., 1982; Selvaraj et al., 1987; Springer, 1990). CD2 promotes the initial stages of T cell contact with a cognate partner such as an APC even prior to T cell receptor (TCR) recognition of a peptide antigen bound to a major histocompatibility complex (MHC) molecule (Koyasu et al., cellular plasma membranes are in contact and thus, offering a greater opportunity for an individual TCR to locate an MHC molecule complexed with a specific antigenic peptide. While it is known that the monomeric affinity of the CD2-CD58 interaction is low (Kd=xcexcM), coligation of CD2 and CD58 molecules on opposing cells within a conjugate pair induces CD2 redistribution to the region of cell-cell contact, resulting in a substantial avidity boost (Arulanandam et al., 1993a; Koyasu et al., 1990; Li et al., 1996; Recny et al., 1990; Sayre et al., 1989).
CD2 functions as a signaling molecule in a number of important settings. First, CD58 interaction with CD2 augments IL-12 responsiveness of activated T cells with regard to proliferation and IFN-xcex3 production (Gollob et al., 1996; Gollob et al., 1995; Wingren et al., 1993). Second, the CD2-CD58 interaction has been reported to reverse T cell anergy (Boussiotis et al., 1994). Third, the CD2 cytoplasmic tail is required for optimal CD2 augmentation of antigen-triggered T cell responses (Hahn and Bierer, 1993; Moingeon et al., 1989b). Hence, in addition to augmenting antigen recognition via its role in adhesion, CD2 facilitates antigen recognition via signal transduction. Consistent with this notion is the fact that T cell proliferation to suboptimal concentrations of anti-CD3 mAb is augmented by CD2 crosslinking through addition of specific pairs of anti-CD2 mAbs (Bierer et al., 1988; Yang et al., 1986). Furthermore, certain pairs of anti-CD2 mAbs can stimulate calcium flux, IL-2 production and cytolytic activity in and of themselves (Meuer et al., 1984; Siliciano et al., 1985).
The molecular basis of CD2-mediated signal transduction is essentially unknown. Truncation and mutation of the CD2 cytoplasmic tail indicate that it is involved in the T cell activation process (Bierer et al., 1988; Chang et al., 1989; Hahn and Bierer, 1993). However, there is no requirement for the CD2 cytoplasmic tail in CD2-mediated cell adhesion and ligand (CD58)-induced CD2 reorganization which occurs subsequent to T cell-APC conjugate formation (Koyasu et al., 1990; Li et al., 1996). A cytoplasmic tail deletion mutant of CD2 lacking the carboxy terminal 92 aa is still capable of relocalizing to the area of cell-cell interaction (Li et al., 1996). These studies do not rule out the possibility that the CD2 cytoplasmic tail is involved in the subsequent regulation of the adhesion complex. CD2-CD58 driven conjugate formation between T lymphocytes and APCs facilitates TCR-mediated antigen recognition of peptide/MHC ligands and subsequently, the attendant downstream cellular activation events. For effective T lymphocyte function, the nature and duration of these cellular events require tight regulation; however, the mechanisms regulating those processes are yet to be elucidated. Moreover, the lack of protein kinase or phosphatase domains or defined signaling motifs within the cytoplasmic tail has complicated the effort to understand the basis for CD2 signaling.
This invention pertains to an isolated CD2BP1 protein, or an active derivative or fragment thereof having CD2BP1 protein activity. In particular embodiments, the CD2BP1 protein is a derivative possessing substantial sequence identity with the endogenous CD2BP1 protein; in other embodiments, the CD2BP1 protein is the long isoform, CD2BP1L protein (SEQ ID NO:1) or the short isoform, CD2BP1S protein (SEQ ID NO:2). The CD2BP1 protein is isolated, that is, purified to homogeneity or is substantially free of other proteins.
The invention also pertains to an isolated nucleic acid molecule which encodes CD2BP1 protein, or an active derivative or fragment thereof having CD2BP1 protein activity, or the complement of said nucleic acid molecule. In one embodiment, the isolated nucleic acid molecule encodes a derivative of CD2BP1 protein possessing substantial sequence identity with the endogenous CD2BP1 protein. In particular embodiments, the isolated nucleic acid molecule encodes a CD2BP1 protein with the same amino acid sequence as endogenous CD2BP1 protein (e.g., CD2BP1L protein or CD2BP1S protein). In another embodiment, the isolated nucleic acid molecule has the same nucleotide sequence as the endogenous gene encoding CD2BP1 protein.
The invention also relates to DNA constructs comprising the nucleic acid molecules described herein operatively linked to a regulatory sequence; to recombinant host cells, such as bacterial cells, fungal cells, plant cells, insect cells and mammalian cells, comprising the nucleic acid molecules described herein operatively linked to a regulatory sequence; and to methods for preparing CD2BP1 protein, by culturing such recombinant host cells. The invention also pertains to an antibody, or an antigen-binding fragment thereof, which selectively binds to CD2BP1 protein or an active derivative or fragment thereof; in a particular embodiment, the antibody is a monoclonal antibody. The invention also relates to a method for assaying for the presence of a CD2BP1 protein in a cell, e.g., in a tissue sample, by contacting the cell with an antibody which specifically binds to CD2BP1 protein.
The present invention also relates to an assay for identifying agents which modulate (inhibit or enhance) the activity of CD2BP1 protein, comprising contacting a composition comprising the CD2BP1 protein, or an active derivative or fragment thereof, with an agent to be tested; and identifying modulation or alteration (e.g., inhibition or enhancement) of CD2BP1 protein activity. For example, a cell or fraction thereof containing CD2BP1 protein, or an active fragment or derivative thereof, can be contacted with an agent to be tested, and the level of CD2BP1 protein activity can be assessed. Novel agents identified by the assays described herein are also included in the invention, as are methods of modulating (inhibiting or enhancing) the activity of CD2BP1 protein by contacting the protein with agents that inhibit or enhance the activity of the protein.
The invention further relates to methods of identifying agents which modulate signal transduction, cell adhesion or cell motility, by identifying modulation of CD2BP1 protein activity; modulation of CD2BP1 protein activity is indicative of an agent that modulates signal transduction, cell adhesion or cell motility. Methods of modulating signal transduction or cell adhesion, by contacting CD2BP1 protein with agents that modulate CD2BP1 protein activity, are also included in the invention. The invention additionally relates to methods of targeting proteins such as kinases or phosphatases to a CD2 molecule in a cell, by linking the protein to be targeted with CD2BP1 protein. It has been shown that association of CD2BP1 with the cytoplasmic tail of CD2 downregulates CD2-based cell adhesion and motility. Thus, cell adhesion and motility can be modulated (inhibited or enhanced) using agents identified by the methods described herein.